An optimized Protocol for Human M2 Macrophages using M‐CSF and IL‐4/IL‐10/TGF‐β Yields a Dominant Immunosuppressive Phenotype

Abstract

Monocytes are highly abundant circulatory effector cells and play a vital role in driving or resolving inflammatory processes depending on their activation phenotype. We investigated and compared a panel of polarization protocols of blood-derived monocytes to achieve a stable, optimal and effective regimen for in vitro induction of immunosuppressive human macrophages, evaluating their surface receptor expression, cytokine profile, scavenging function and ability to suppress T-cell proliferation. Importantly, we assessed the effect of copolarization or secondary pro-inflammatory stimulation of a primary anti-inflammatory activation phenotype. A combination of IL-4/IL-10/TGF-β yielded a relatively stable and dominant immunosuppressive phenotype characterized by higher IL-10 production and down-regulated TNF-α, IL-6, CD86, CD274 and MHC II expression. Functionally, IL-4/IL-10/TGF-β-stimulated macrophages (M2) had a potent deactivating effect on a subsequent pro-inflammatory LPS/IFNγ-activated macrophage (M1) stimulation and significantly suppressed T-cell proliferation. Monocytes derived from patients with chronic inflammatory diseases could be induced to be anti-inflammatory using this protocol. Pre-differentiation with GM-CSF or M-CSF was further demonstrated to enhance final M1/M2 activation status. Our findings indicate a robust polarization protocol for generation of specific immunosuppressive human monocyte-derived macrophages.

Dietary Consumption of Black Raspberries or Their Anthocyanin Constituents Alters Innate Immune Cell Trafficking in Esophageal Cancer

“…Further, MIP-1a and IFNg, cytokines secreted by NK cells (20,48), were expressed at significantly more in the plasma of rats fed BRBs, ACs, or PCA at weeks 15 and 25, as well. This suggests that BRBs, ACs, and PCA may increase NK-cell activity in NMBA-induced esophageal tumorigenesis in rats.…”

“…This macrophage subtype is associated with a more aggressive form of esophageal SCC in humans (26). Because M2 macrophages are generally anti-inflammatory (20), higher numbers in esophageal tissue may inhibit the activity of other killer immune cells, thus preventing immune cell-mediated tumor cell destruction (27). BRB ACs and PCA can downregulate the expression of IL4 by rat innate immune cells in vitro (28).…”

“…We reported that dietary intake of BRBs, BRB ACs, or PCA increases PTX3 expression in NMBA-treated rat esophagi, suggesting that whole berries and their constituents may reduce neutrophil trafficking within the esophagus. In addition, cytokines IL1b and IL6, associated with the proinflammatory macrophage subtype M1 (18)(19)(20), are overexpressed in human esophageal SCC and are associated with a poor prognosis (21,22). BRBs may be protective here through reduction of inflammation in humans by inhibiting IL1b expression (23).…”

Generation of anti-inflammatory macrophages for implants and regenerative medicine using self-standing release systems with a phenotype-fixing cytokine cocktail formulation

“…The use of multiple factors for macrophage polarization is also physiologically relevant since the cells in in vivo conditions are always influenced by complex cytokine milieu. Double and triple combinations of anti-inflammatory factors IL4, IL10 and TGFβ1 were previously shown to be more efficient than single cytokines, and induced robust tolerogenic phenotype in mouse and human macrophages [8,9,12]. In addition, macrophages stimulated by such cytokine combinations partially retained anti-inflammatory phenotype in vivo and their adoptive transfer protected mice from chronic inflammatory disorders including autoimmune type 1 diabetes and adriamycin nephrosis [8,9].…”

“…This has led us to hypothesize that a designed cytokine cocktail might be more potent in establishing and more importantly fixing a desired cytokine phenotype than a single cytokine induction. Recently, human monocyte-derived macrophages with dominant anti-inflammatory phenotype were generated using cytokine cocktail consisting of M-CSF (50 ng/ml), IL4 (20 ng/ml), IL10 (20 ng/ml) and TGFβ1 (20 ng/ml) in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) [12].…”